Single-Cell Genome and Group-Specific dsrAB Sequencing Implicate Marine Members of the Class Dehalococcoidia (Phylum Chloroflexi) in Sulfur Cycling

Abstract

The marine subsurface sediment biosphere is widely inhabited by bacteria affiliated with the class Dehalococcoidia (DEH), phylum Chloroflexi, and yet little is known regarding their metabolisms. In this report, genomic content from a single DEH cell (DEH-C11) with a 16S rRNA gene that was affiliated with a diverse cluster of 16S rRNA gene sequences prevalent in marine sediments was obtained from sediments of Aarhus Bay, Denmark. The distinctive gene content of this cell suggests metabolic characteristics that differ from those of known DEH and Chloroflexi The presence of genes encoding dissimilatory sulfite reductase (Dsr) suggests that DEH could respire oxidized sulfur compounds, although Chloroflexi have never been implicated in this mode of sulfur cycling. Using long-range PCR assays targeting DEH dsr loci, dsrAB genes were amplified and sequenced from various marine sediments. Many of the amplified dsrAB sequences were affiliated with the DEH Dsr clade, which we propose equates to a family-level clade. This provides supporting evidence for the potential for sulfite reduction by diverse DEH species. DEH-C11 also harbored genes encoding reductases for arsenate, dimethyl sulfoxide, and halogenated organics. The reductive dehalogenase homolog (RdhA) forms a monophyletic clade along with RdhA sequences from various DEH-derived contigs retrieved from available metagenomes. Multiple facts indicate that this RdhA may not be a terminal reductase. The presence of other genes indicated that nutrients and energy may be derived from the oxidation of substituted homocyclic and heterocyclic aromatic compounds. Together, these results suggest that marine DEH play a previously unrecognized role in sulfur cycling and reveal the potential for expanded catabolic and respiratory functions among subsurface DEH.

Importance: Sediments underlying our oceans are inhabited by microorganisms in cell numbers similar to those estimated to inhabit the oceans. Microorganisms in sediments consist of various diverse and uncharacterized groups that contribute substantially to global biogeochemical cycles. Since most subsurface microorganisms continue to evade cultivation, possibly due to very slow growth, we obtained and analyzed genomic information from a representative of one of the most widespread and abundant, yet uncharacterized bacterial groups of the marine subsurface. We describe several key features that may contribute to their widespread distribution, such as respiratory flexibility and the potential to use oxidized sulfur compounds, which are abundant in marine environments, as electron acceptors. Together, these data provide important information that can be used to assist in designing enrichment strategies or other postgenomic studies, while also improving our understanding of the diversity and distribution of dsrAB genes, which are widely used functional marker genes for sulfur-cycling microbes.

FIG 1

Phylogenetic analysis of the 16S rRNA gene from DEH-C11 in comparison to those of other members of the class DEH. The tree is based on the maximum likelihood algorithm. Branches in orange highlight the DSC-GIF3-B subgroup, to which the 16S rRNA gene of DEH-C11 (leaf label highlighted in red) is affiliated. Branches in green highlight the clade containing all known organohalide-respiring phylotypes, and leaf labels of sequences from cultivated bacteria or sequences implicated in organohalide respiration are highlighted in green. Branches in blue highlight the GIF9-A clade. Leaf labels in blue highlight sequences for which genomic information is available (18–20). The scale bar represents 5% sequence divergence. PCB, polychlorinated biphenyl; PCE, tetrachloroethene; SIP, stable isotope probing.

FIG 2

A simplified schematic of putative biochemical properties predicted from genomic content of DEH-C11. The redox cycling schematic for sulfite reduction is based on model proposed by Venceslau et al. (24). Aromatic substrates depicted are (left to right) phenylalanine, phenol, and pyrogallol. The heterocyclic compound depicted is creatinine. Nuo, NADH:ubiquinone oxidoreductase complex; TRAP, tripartite ATP-independent periplasmic transporters; DMSO, dimethyl sulfoxide; DMS, dimethyl sulfide; Dsr, dissimilatory sulfite reductase; S-layer, surface-layer protein coat.

FIG 3

Representation of the gene order present on the contig (IDBA scaffold 11) containing the dissimilatory sulfite reductase operon and associated genes from DEH-C11 (top) and D. lykanthroporepellens BL-DC-9 (bottom). Gene names in blue denote that the best BLASTP hits were to D. lykanthroporepellens BL-DC-9 or other Chloroflexi strains. Blue shaded lines between the two gene order representations show regions with high sequence similarity and synteny determined by tBLASTx as implemented using EasyFig (93). Degrees of tBLASTx sequence identity are depicted in the colored legend. The red blocks at the end of the DEH-C11 contig representation denote the ends of the contig sequence. The red and blue lines within the genes of the DEH-C11 represent the regions of the genome amplified by long-range PCR.

FIG 4

Phylogenetic analysis of DsrAB from DEH-C11 (highlighted in red) and cloned DsrAB sequences as determined by the evolutionary placement algorithm (88), which was used to place sequences onto a previously constructed DsrAB consensus tree (34). Orange leaf labels indicate sequences retrieved by long-range PCR in this study. L.A.-Firmicutes, DsrAB proteins that were laterally acquired by certain Firmicutes species of the Deltaproteobacteria (34). The red dots on leaf labels indicate that the DsrAB sequences are derived from amplified sequences that were sequenced by primer walking and with pentanucleotide signatures similar to that of the DEH-C11 genome (see Fig. S1 in the supplemental material). The grey scale bar represents 5% sequence divergence.

FIG 5

Phylogenetic analysis of RdhA derived from DEH-C11 (highlighted in red), DEH-derived RdhA retrieved from metagenomes, and RdhA from pure reference strains. The tree is based on the maximum-likelihood algorithm. Branches of metagenome-derived RdhA sequences determined to originate from DEH- or Chloroflexi-related contigs are highlighted in orange (50, 51, 74). Branches corresponding to the “conserved syntenic” Dehalococcoides species RdhA are highlighted in dark blue, while the Dehalococcoidales RdhA clade are highlighted in light blue. Nearly full-length RdhA sequences from the Smkt-3 clade (two of six available were nearly full length) from subsurface sediments of Shimokita peninsular were included (49). Branches of RdhA derived from aerobic bacteria are highlighted in purple. The grey scale bar represents 20% sequence divergence. WOR, White Oak River.